Navigating to the PCR Tab
All primer related features can be found under the “PCR” tab in the top banner. Once here you can find the “Add”, “Edit”, “Copy” and “Remove” buttons which are your main primer editing tools.
Adding and Removing Primers in the Sequence
To add primers directly you can use the “Add” button in the “PCR” tab. If you have not selected a region in your sequence the “Add primer” modal box will prompt you to input the Name and the Sequence of the primer. Once you have input the sequence you will notice it is displayed in green, and the primer binding site is shown in a preview below. It is also possible to add 5’ phosphorylation via the “Select modification” dropdown or to add restriction sites.
If you have already selected a region of your sequence and use the “Add” button to add primers, you will see the dropdown option to select the which strand the primer will bind on and can choose between “Use forward strand” and “Use reverse strand”.
You will notice that the modal for adding primer is identical, the only difference is that the “Sequence” if preselected will be displayed in black letters instead of green. When you are finished with designing your primers select the “Save” button and the primer will be saved to your sequence file and will be visible if primers are toggled on in the “View” menu in the top banner.
It is also possible to add primers by right clicking the sequence and selecting “Create” > “New primer” or via the “View” menu with the “Primers” dropdown. If you select “Add primer” from the dropdown you will open the same “Add primer” modal as above.
To remove primers, start by selecting a primer by clicking on it. Once selected the “Remove” button will become clickable in the top banner under the “PCR” tab. After clicking this you will receive a final warning / check which you can confirm to permanently delete the primer.
Again, the primer delete / remove function is also available via the dropdown in “View” > “Primers” > “Remove primer”. Alternatively, you can use the delete or backspace button on your keyboard, if this method is used you can choose what should be deleted either “Primer and sequence” or “Primer only”.
Editing and Copying Primers
To edit a primer you can simply double click it, this will open the “Edit primer” modal box which is functionally identical to the “New primer” modal. Alternatively, you can right click the primer and select “Edit Primer” or you can select the primer by clicking it and then navigate to the “PCR” tab in the top banner and select “Edit”. Lastly, you can also access primer editing via “View” > “Primers” > “Edit primer”.
When copying primers there are several options the first is to select the primer by clicking on it and then using the “Copy” button under the “PCR” tab in the top banner. This will open the “Add primer” modal where you will see that the primer has been duplicated with the term (copy) appended to the name. This is useful if you are creating several primer variants to test with slight variations on length or position. This copy method can also be found under “View” > “Primers” > “Copy primer”.
The next option for copying primers is by selecting the primer and right clicking it and navigating to “Copy” > “Copy” this will copy the primer and the sequence, when pasting somewhere else in the sequence the primer and sequence will be pasted in the new location. This also transfers features or partial features.
The last copy option is via keyboard shortcut, on Windows you can use Ctrl + C and on MacOS you can use Cmd + C. This works identically to the right click method and copies anything within the selection; if you have selected a primer it will copy the primer and any other features appearing within the selection.
